Most successful attempts to make plants that are resistant to insect attack have been obtained by, and are believed to be dependent on, high level expression of insecticidal toxins (Estruch et al., 1997; Witowsky & Siegfried, 1997). Most particularly for the Bt toxins, which as full-length proteins were found to be expressed poorly in plants, efforts have concentrated on increasing expression of these toxins in plants by use of strong constitutive promoters and by modification of the genes encoding them (Vaeck et al., 1987; Barton et al., 1987). More recently spatial regulation of expression of the insecticidal protein in those tissues susceptible to attack or triggered by feeding of the insect has been suggested as possibly providing advantages for resistance management (Peferoen & Van Rie, 1997). One of the major conditions for obtaining regulatory approval for transgenic insect resistant plants is the availability of an insect resistance management strategy. The currently favored strategy is the combination of 100% toxicity of the transgenic plants to the target pest(s), obtained by high dose expression of a specific toxin and this during the full life-cycle of the pest, with the use of refuges of non-transgenic plants, which allow the maintenance of the target pest population (De Maagd et al. 1999). In order to be able to comply with such a strategy, the use of strong constitutive promoters in the engineering of insect-resistant plants has further been encouraged.
Inducible expression of insect resistance has primarily been examined for the potato proteinase-inhibitor genes (pin1 and pin2), which are part of the natural defense system in plants and upon wounding, ensure the generation of a systemic signal throughout the plants (Green and Ryan, 1972; Hilder et al. 1987). Introduction of the pin2 gene in rice resulted in high-level systemic accumulation of the protein in rice plants, which showed increased resistance to major pests (Duan et al., 1996). Breitler et al. (2001) describes the use of the C1 region of the maize proteinase inhibitor (MPI) gene to drive wound-inducible expression of the cry1B coding sequence in rice and the first transformants were found to effectively protect rice against stemborer attack, the wound-induced expression was described to be both locally and systemically.
In a small-scale laboratory experiment, transgenic cabbage leaves transformed with the cry1Ab3 gene placed under the control of the inducible vspB promoter from soybean were as toxic to diamondback moth as those transformed with the same gene under control of the 35S promoter, but wound-inducibility was not demonstrated (Jin et al., 2000).
The TR2′ promoter of the mannopine synthase gene of Agrobacterium tumefaciens, originally considered to direct constitutive expression (Velten et al. 1984; Vaeck et al. 1987), has been used to direct wound-inducible expression of a native Cry1Ab gene in tomato, which led to relatively low expression and only moderate insect control (Reynaerts & Jansens, 1994). Though a possible broad application for expression of Bt proteins has been suggested (Peferoen, 1997), there appear to be discrepancies between the reports on the expression pattern of the TR2′ promoter in tobacco and other dicots (Ni et al. 1995). In general, the use of monocot promoters is preferred for optimal expression of genes in monocots (Shimamoto, 1994) and certain promoters have been found to have different cis-acting elements in monocots and dicots (Luan et al. 1992). The contribution of different elements of the TR2′ promoter on its activity was investigated in maize protoplasts (Fox et al., 1992), but there have been no reports on the expression pattern of the TR2′ promoter in a monocotyledonous plant. Furthermore, the strongest deletion mutant was found to be 20 times less active than the CaMV 35S promoter (Fox et al., 1992).
Agrobacterium mannopine synthase promoters have been characterized as being constitutive, root-specific, and tissue-specific promoters, e.g., see U.S. Pat. Nos. 6,291,745, 6,320,100 and 6,313,378. In U.S. Pat. No. 5,641,664, it is believed that various constitutive and organ- and tissue-specific promoters that are used to direct expression of genes in transformed dicotyledonous plants will also be suitable for use in transformed monocots. As part of a general list of suitable foreign constitutive promoters for transforming plant cells in this patent are the TR1′ and the TR2′ promoters which drive the expression of the 1′ and 2′ genes, respectively, of the T-DNA of Agrobacterium, and are said to be wound-induced promoters. This patent shows no monocot plant transformed with the TR2′ or TR1′ promoter, nor is there any suggestion that these promoters are wound-induced promoters in monocot plants or are useful for expression of an insecticidal protein in a monocot plant.
The present invention describes how the TR2′ promoter can be used to direct wound-induced expression of an insecticidal protein in monocot plants to obtain insect resistance. Such wound-inducible expression of the TR2′ promoter leads to a strong but localized increase of expression of the insecticidal protein. The putative effect on plant vigor and growth, observed with high level expression of some Bt proteins particularly upon repeated inbreeding (such as reported in WO 00/26378 for Cry2Ab) is likely to be reduced as the limited expression of the protein should minimize any burden on functions important for maintaining the agronomic qualities of the engineered crop. This is also an important factor when stacking of different traits (or different Bt proteins) is envisaged. As high-dose expression levels are attained upon contact with the target pest, such plants should comply with current IRM strategies. Additionally, the combination of the specificity of the insect toxin produced with a spatially and temporally limited expression pattern (i.e. in tissues susceptible to wounding, upon wounding) is likely to reduce exposure of non-target organisms, which can be considered advantageous over constitutive production of the toxin by the plants. Thus, an effective system of regulated expression ensuring effective insect resistance for major crops such as corn and rice is of interest from both a regulatory and agricultural point of view.